Radiolabeled herbicides binding

Herbicides that inhibit photosynthetic electron flow prevent reduction of plastoquinone by the photosystem II acceptor complex. The properties of the photosystem II herbicide receptor proteins have been investigated by binding and displacement studies with radiolabeled herbicides. The herbicide receptor proteins have been identified with herbicide-derived photoaffinity labels. Herbicides, similar in their mode of action to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) bind to a 34 kDa protein, whereas phenolic herbicides bind to the 43-51 kDa photosystem II reaction center proteins. At these receptor proteins, plastoquinone/herbicide interactions and plastoquinone binding sites have been studied, the latter by means of a plastoquinone-deriv-ed photoaffinity label. For the 34 kDa herbicide binding protein, whose amino acid sequence is known, herbicide and plastoquinone binding are discussed at the molecular level. 

In 1979, the concept of a photosystem II herbicide binding protein with different but overlapping binding sites for the various photosystem II herbicides was simultaneously established by Trebst and Draber ( 5) and Pfister and Arntzen (6). This idea of a herbicide receptor protein proved to be extremely fruitful because the techniques of receptor biochemistry were now applicable. Tischer and Strotmann (7) were the first investigators to study binding of radiolabeled herbicides in isolated thylakoids. 

As already stressed, photosystem II herbicides bind reversibly to their binding site. A1tough radiolabeled herbicides are available, it is impossible to identify the herbicide receptor protein without a chemical modification of the herbicide that allows for covalent... 

In conclusion, observations made in the last few years, especially the binding studies with radiolabeled herbicides, the photoaffinity labeling technique, and the advances of molecular biology have substantially added to our knowledge of the mechanism of action of photosynthetic herbicides. However, many questions also remain to be answered. 

Herbicide Binding Assays. Control and trypsin-treated chloroplast thylakoids were suspended in PSNM buffer. Buffer (1 ml volume) containing 50 Chi was incubated 3 min with l C-atrazine (specific activity 27.2 pCi/mg). Chloroplasts were pelleted and an aliquot of the supernatant was removed for determination of the amount of unbound atrazine. Details of this procedure are described elsewhere (6, 9). Radiolabeled atrazine was a gift of Dr. H. LeBaron, CTBA-GEIGY, N. Carolina. 

The distinctly different behavior of the phenol-type herbicides following trypsin treatment suggests that different determinants within the PS II protein complex establish the "domains" that regulate the binding properties of these inhibitors. In spite of the fact that phenol-type herbicides will displace bound radiolabeled herbicides such as diuron, these inhibitors show noncompetitive inhibition (29, 30). At present, there are three lines of evidence which favor TH e involvement of two domains within the PS II complex that participate in creating the binding sites for these herbicides (a) isolated PS II particles can be selectively depleted of a polypeptide with parallel loss of atrazine sensitivity, but not dinoseb inhibition activity (33) (b) in resistant weed biotypes, chloroplast membranes that exhibit extreme triazine resistance have increased sensitivity to the phenol-type herbicides (13) and (c) experiments with azido (photoaffinity) derivatives of phenol and triazine herbicides result in the covalent labeling of different PS II polypeptides (, 31). 

Herbicide binding — The amount of radiolabelled herbicide bound to membranes was followed at different illumination times and the activity expressed as per cent of initial value. 

Many inhibitors of photoinduced electron transport, including a large number of herbicides, have been shown to bind reversibly and competitively to the protein. The studies volve displacement of a radiolabeled inhibitor, such as [ C]-atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)- -trlazine], by an uy abeled inhibitor (29). Only marginal displacement of [ Cj-atrazine was observed by high concentrations of umbelliferone and naringenin. The other allelochemicals produced no measurable effects. 

Surugiu et al. [67] have introduced an Enzyme Immuno-Like Assays (EzILA) for the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The label was a 2,4-D conjugate with the tobacco peroxidase (TOP) enzyme, which allows for both colorimetric and chemiluminescent detection. In this case, the polymer imprinted with 2,4-D was synthesized in the form of microspheres. In contrast, despite its higher binding capacity for radiolabeled 2,4-D, a conventional MIP prepared by bulk polymerization showed only weak binding of the 2,4-D-TOP tracer. 

Recently, Schloss et al (33) showed that IM and TP were able to quantitatively displace a radiolabelled SU herbicide from ALS, indicating competitive binding. Curiously, the SU ligand was also displaced by the quinone, Qo. It was proposed that SU, TP, and IM bind to ALS in a vestigial quinone binding site associated with the evolution of ALS from pyruvate oxidase. This enzyme is an FAD-protein that catalyzes the oxidation of pyruvate to acetate. 

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